Method and apparatus for rapid biohydrogen phenotypic screening of microorganisms using a chemochromic sensor

ABSTRACT

The invention provides an assay system for identifying a hydrogen-gas-producing organism, including a sensor film having a first layer comprising a transition metal oxide or oxysalt and a second layer comprising hydrogen-dissociative catalyst metal, the first and second layers having an inner and an outer surface wherein the inner surface of the second layer is deposited on the outer surface of the first layer, and a substrate disposed proximally to the outer surface of the second layer, the organism being isolated on the substrate.

CROSS REFERENCE TO RELATED APPLICATIONS

This 35 U.S.C. 111(a) application is filed pursuant to 35 U.S.C.119(e)(1) for an invention disclosed in U.S. Provisional Application No.60/086,313 entitled: A CHEMOCHROMIC SENSOR FOR RAPID BIOHYDROGENPHENOTYPIC SCREENING, filed May 21, 1998.

CONTRACTUAL ORIGIN OF THE INVENTION

The United States Government has rights in this invention pursuant toContract No.DE-AC36-98GO10337 between the United States Department ofEnergy and the Midwest Research Institute.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to chemochromic sensor films for use in rapidlyscreening isolate organisms capable of producing hydrogen.

2. Description of the Prior Art

Light-induced biological hydrogen production represents a potentiallycost-effective system for the production of renewable non-pollutingenergy. Photobiological hydrogen production is catalyzed by nitrogenaseand hydrogenase enzyme systems that are present in bacteria,photosynthetic bacteria, cyanobacteria, and green algae. Green algae,such as Chlamydomonas reinhardtii, can photoevolve hydrogen only throughan inducible, reversible hydrogenase enzyme using water as the electronsource. While hydrogenase-catalyzed hydrogen photo-production haspotential as an efficient energy source, one of the major obstacleswhich currently limits any commercial application of this process is thedeactivation of the hydrogenase enzyme in the presence of oxygen,produced in the water-splitting process of photosynthesis. Thus, it isdesirable to isolate and select for bacterial, or algal mutant organismswhich exhibit an oxygen-tolerant hydrogen production phenotypicresponse, in order to further the commercial production of light-inducedhydrogen.

Two recent methods have been used in order to select for C. reinhardtiimutants which exhibit the oxygen tolerant hydrogen production phenotypicresponse. These methods are based on either the hydrogen production orhydrogen uptake activity of the reversible hydrogenase enzyme. The firstapproach is dependent on the ability of algal cells to produce hydrogenin competition with a drug that, when reduced, releases products toxicto the cells. Hydrogen production selective pressure is applied in thepresence of increasing oxygen-stress to enrich for oxygen-tolerantorganisms. The second approach is based on algal cell growth, usinghydrogen as an electron source for carbon dioxide fixation. The additionof oxygen during the hydrogen uptake selection is then used to selectfor oxygen-tolerant organisms.

The evolution of hydrogen by an organism has been assayedamperometriclly. The amperometric determination uses a Clark typeelectrode that can be biased versus Ag/AgCl to determine either thehydrogen or oxygen concentration present in an assay chamber. Algalcells are induced under anaerobic conditions prior to the measurements.The algae are then anaerobically injected into an assay chambercontaining an assay buffer (50 mM MOPS, pH 6.8), which is pre-adjustedto different initial concentrations of oxygen. The cells are thenincubated in the dark and illuminated with a saturating heat-filteredincandescent light to induce hydrogen evolution. Initial hydrogenevolution rates are derived from the initial slopes of each curve, andgas concentrations are corrected for the decrease in aqueous solutionsolubility. A gas chromatograph can also be used to assay for hydrogenproduction capacity. However, these assays for oxygen-tolerant hydrogenphoto-production are very costly, time consuming, and have been theprime rate limiting factor in the rapid identification and selection ofmore desirable mutant organisms.

Hunter, in U.S. Pat. No. 5,668,301 discloses a hydrogen sensitive metalalloy that contains palladium and titanium to provide a larger change inelectrical resistance when exposed to the presence of hydrogen. Thealloy is deposited on a substrate and a thin film is connected acrosselectrical circuitry to provide a sensor device that can be used forimproved sensitivity and accuracy of hydrogen detection.

U.S. Pat. No. 5,367,283 issued to Lauf et al., discloses a thin-filmhydrogen sensor element comprised of an essentially inert,electrically-insulating substrate having a thin-film metallizationdeposited thereon which forms at least two resistors on the substrate.The metallization comprises a layer of Pd or a Pd alloy for sensinghydrogen and an underlying intermediate metal layer for providingenhanced adhesion of the metallization to the substrate. The differencein electrical resistances of the covered resistor and uncovered resistoris related to the hydrogen concentration in a gas to which the sensorelement is exposed.

U.S. Pat. No. 4,324,761, issued to Harris, discloses a hydrogen detectorcomprised of a substrate supporting a electrically conducting base metalfilm, and upper electrically conducting diffusion barrier metal film, apolycrystalline film of titanium dioxide sandwiched between the base anddiffusion barrier films, the polycrystalline titanium dioxide filmelectrically insulates the base film from the diffusion barrier film,the base film, being in electrical contact with the titanium dioxidefilm, an insulating layer electrically insulating the titanium dioxidefilm from the diffusion barrier film except for a predetermined surfaceportion thereof in electrical contact with the diffusion barrier film;wherein the predetermined electrically contacting portion issufficiently large to produce a measurable electrical conductance, anelectrically conducting or non-conducting catalytic top film of metalable to dissociate hydrogen into its atomic form in electrical contactwith the diffusion barrier film and a least coextensive with the barrierfilm throughout the predetermined electrically contacting portion, ascan best be seen in the cross-sectional view of FIG. 2.

U.S. Pat. No. 4,324,760 to Harris, disclosed a hydrogen detector havinga substrate supporting an electrically conducting base metal film, anelectrically conducting top film of metal able to dissociate hydrogeninto atomic form, a polycrystalline film of titanium dioxide sandwichedbetween the base and top films, wherein the polycrystalline titaniumdioxide film electrically insulates the base film from the top film, thebase film being in electrical contact with the titanium dioxide film, aninsulating layer electrically insulating the titanium dioxide film fromthe top film except for a predetermined surface portion thereof inelectrical contact with the top film, and wherein the predeterminedelectrically contacting portion is sufficiently large to produce ameasurable electrical conductance that varies with the concentration ofhydrogen in the atmosphere surrounding it.

Each of the foregoing patents issued to Hunter, Lauf, et al., and Harrisdisclose electrical devices, which generate an electrical signal,determinative of the presence of hydrogen. However, these devices do notspatially resolve the point where the gas in produced in relation to thesample surface. Therefore, these devices would not be useful todiscriminate the specific location of a colony which produces hydrogengas where the sample, to be screened, consists of many colonies of theorganism.

U.S. Pat. No. 3,567,383 issued to Langley et al. discloses a detectorfor hydrogen having as its sensing device a thin film comprised ofpalladium or platinum oxide, which oxide on contact with hydrogenreduces to the corresponding metal. The differences in properties,electrical or optical, of the oxide and metal film are used to detectthe presence of hydrogen. While this patent provides an optical meansfor detecting hydrogen which could provide spatial resolution it doesnot provide the sensitivity (0.02% hydrogen concentration), rapidresponse rate (a few seconds), and economy in manufacture which aredesirable for rapidly screening colonies of hydrogen-producing organismson a substrate.

The development of a rapid screening method and device for the detectionof hydrogen-producing mutants would greatly enhance the analysis of thesurvivors of selective pressure methods, in the rapid isolation ofdesirable mutant organisms. Furthermore, a rapid screening would assistin the analysis of mutants derived through a molecular biologicalapproach to further the oxygen-tolerance of the hydrogenase enzyme.

Therefore, what is needed, is a rapid screening method for theoxygen-tolerant, light-induced hydrogen phenotype in mutant organisms.

SUMMARY

It is therefore an object of the present invention to rapidly screen forisolate microorganisms which exhibit an oxygen-tolerant hydrogenphenotype.

It is another object of the present invention to use a chemochromic filmto detect isolate mutant colonies of green algae, which produce hydrogenunder either anaerobic or aerobic conditions.

It is yet another object of the present invention to develop a screeningassay for isolating individual oxygen-tolerant, hydrogen-producing,mutant green algae colonies having an assay sensitivity limit of atleast a 4 nmoles of hydrogen evolved per colony of microorganism, to bescreened.

Briefly, the invention provides a system for identifying ahydrogen-gas-producing organism, comprising a sensor film having a firstlayer comprising a transition metal oxide or oxysalt and a second layercomprising a hydrogen-dissociative catalyst metal, the first and secondlayers having an inner and an outer surface wherein the inner surface ofthe second layer is deposited on the outer surface of the first layer,and a substrate adjacent to the outer surface of the second layer, theorganism located on the substrate.

Unless specifically defined otherwise, all technical or scientific termsused herein have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Although anymethods and materials similar or equivalent to those described hereincan be used in the practice or testing of the present invention, thepreferred methods and materials are now described.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a side view of the preferred embodiment of the screeningassay.

FIG. 2 is a cross-section of a preferred embodiment of the sensor filmlayer system.

FIG. 3A is a top view of an agar plate having surviving green algaemutant colonies obtained through selective pressure.

FIG. 3B shows the sensitization reaction. The chemochromic film ispositioned over the filter paper, and the algae are illuminated withsaturating light to induce hydrogen production.

FIG. 3C shows a color change in the chemochromic film (note the roundspots), which spatially identifies the isolated colonies that producehydrogen. The chemochromic film has been removed from the agar place andhas been placed on a white background.

FIG. 3D, shows the reversible nature of the color reaction afterexposure of the film to air for 5 minutes, following the sensitizationreaction, and hence the confirmation that hydrogen was in fact detected.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is a very sensitive chemochromic sensor film, andmethod for using the film, to detect the production of a gas in thepresence of an isolate colony of a microorganism. The device comprises achemochromic film placed in a spaced relationship to an isolate colonyof a microorganism. The film exhibits a reversible color change, at apoint just over the gas-producing colony, which varies in intensitydepending on the concentration of gas produced. The color change occursunder either anaerobic or aerobic conditions.

In a preferred embodiment, the gas to be detected is hydrogen. Thechemochromic film uses a layer system, having transition metal oxides oroxysalt 7 and a hydrogen dissociative catalyst metal 6, wherein thetransition metal oxide or oxysalt 7 is selected from the groupconsisting of WO₃, Nb₂O₃ and CoMoO₄, and the hydrogen dissociativecatalyst metal is selected from the group consisting of platinum,rhodium and palladium. The layer can further comprise a fluorinatedhydrocarbon polymer 8 wherein the fluorinated hydrocarbon polymercomprises TEFLON. The chemochromic film is normally transparent. In asensitization reaction, when hydrogen gas reacts with the tungstentrioxide, it causes a reversible blue color change which grows inintensity as the concentration of hydrogen increases. The hydrogendissociative catalyst metal serves to accelerate this reaction. Whenhydrogen gas is removed from the vicinity of the film, hydrogen in thefilm diffuses to the film surface and escapes as hydrogen gas or isoxidized to water if oxygen is present, and the film returns to it'stransparent state for reuse.

In another preferred embodiment a grid system, and a piece of filterpaper are located over several colonies, growing on agar in a Petridish, for phenotypic screening. When small quantities of hydrogen areproduced in the vicinity of the film, the grid location of the sensorfilm blue coloration indicates the location of the colony which exhibitsthe oxygen-tolerant hydrogen phenotype. In this way, the sensor helps toidentify which, among many colonies of microorganism is producinghydrogen gas under the selected experimental conditions.

Referring now to the drawing figures, in which like numerals representlike features there is shown in FIG. 1 film 20 is a chemochromic filmlayer, positioned in plastic dish 1, on top of filter paper 15. Onfilter paper 15, grid 35 is positioned to locate isolate microorganismcolony 25, which is growing on agar surface 10, poured into atransparent culture dish 1. Film 20 exhibits a color change at a pointjust over a hydrogen-producing colony 25. Film 20 exhibits the colorchange in the presence of hydrogen either under anaerobic or aerobicconditions, upon sensitization with light source 30.

In FIG. 2, chemochromic film 20 uses a layer system comprising a layerof tungsten trioxide (WO₃) 6, deposited by vacuum thermal evaporationonto glass substrate 5, to a thickness of approximately 500 nm. A verythin layer of palladium 6, approximately 2.2 nm thick, is also depositedby vacuum thermal evaporation on top of the tungsten trioxide layer. Thelayer system can further comprise, as in the preferred embodiment, apolymer layer of TEFLON 8, to a thickness of approximately 100-1000 nm,to inhibit water and other contaminants from reaching the film layer 20,6, and 7. While it is preferred that the deposition of film layer 20, 6,and 7, onto substrate 5, is by vacuum thermal evaporation, it may beaccomplished by any other method, well know in the art, including,without limitation, rf-, and dc-sputtering, and laser ablation.Chemochromic film 20 is transparent when not in the presence ofhydrogen. When hydrogen gas reversibly reacts with the tungstentrioxide, the film changes to a blue color (H_(x)WO₃) which grows inintensity as the concentration of hydrogen (x in the formula, H_(x)WO₃)increases. When hydrogen gas is displaced from the vicinity of film 20,the hydrogen in the H_(x)WO₃ diffuses to the surface of film 20 andescapes either as hydrogen gas, or is oxidized to water, if oxygen ispresent.

When hydrogen is produced, in small quantities, near the sensor coating,as for example with colonies of green algae, the local coloration of thefilm, as outlined within the grid, identifies the location of thehydrogen-producing colony. In this way, the film helps to identifywhich, among many plated colonies of algae are producing hydrogen gasunder experimental conditions.

EXAMPLE

Wild type C. reinhardtii (WT) was obtained from the University ofColorado at Boulder and a cell-wall-less strain of C. reinhardtii (cw15)was obtained from the Chlamydomonas Genetics Center, Duke University.Algal cell suspensions were grown photoautotrophically either in Sager'sminimal medium (WT), or in a modified Sueoka'a high salt medium, asdescribed in Ghirardi et. al., Development of an Efficient AlgalH₂-Producing System, Proceedings of the 1996 U.S. DOE Hydrogen ProgramReview, Vol. I, 285-302, (1997). Suspension cultures were grown underillumination at 25° C., using cool white fluorescent lights (8 W/m²),and agitated with a bubble mixture of 1.7% CO₂ in air. Plated colonieswere prepared by centrifugation of suspension cultures. Harvesting ofcell suspensions was done at 2000 g, for 10 minutes, and resuspendedcells were inoculated on either 1.5% (WT), or 0.8% (cw15) agar gel insterile plastic Petri dishes.

Mutageneses

In order to generate C. reinhardtii mutants to be used in the twoselective pressure methods described above, the cultures were exposed tonitrosoguanidine (“NTG”), a chemical mutagen that induces random pointmutations in algae, according to the method of Harris E., TheChlamydomonas Sourcebook, Academic Press, New York (1989). The randompoint mutations were induced in an algal cell suspension (3×10⁶/ml), incitrate buffer (0.025 M sodium citrate, pH 5.0), with 1 μg/ml NTG for 30minutes, in the dark. We had previously determined that this inductionprotocol kills 50% of the cell-wall-less cells in suspension. The cellsuspensions were then washed to remove any residual NTG, suspended ingrowth medium, and incubated for two to three days, under illumination,in order to allow for chromosome segregation and mutational expression.Finally, a dark aerobic starvation of the cells followed, for two tothree days, to deplete the internal cellular storage reserve prior tohydrogen-uptake selective pressure.

Hydrogen-Uptake Selective Pressure

Selection using hydrogen-uptake conditions was first applied by McBrideet al, Mutational Analysis of Chlamydomonas reinhardtii, Application toBiological Solar Energy Conversion, Biological Solar Energy Conversion,77-86, Academic Press, New York, N.Y. (1977), who subjected a populationof WT cells to photoreductive conditions in the presence of controlledoxygen concentrations. Surviving algal cells grew by fixing carbondioxide with electrons obtained from the oxidation of hydrogen (usinghydrogenase hydrogen-uptake activity) and adenosine triphosphategenerated by a cyclic electron transfer around photosystem I.Photosynthetic water oxidation was blocked by the presence of aphotosystem II inhibitor, dicholoromethlyurea (“DCMU”). This selectivepressure has been much more specific for oxygen-tolerant organisms thanfor hydrogen production selection.

Based on the McBride, et al. disclosure, we used an optimized hydrogenuptake selective pressure procedure on the above mutagenized suspension.The hydrogen-uptake selective pressure was applied by treating a liquidculture of the mutagenized wild-type C. reinhardtii cells with a 10-100μM DCMU, or 15 μM DCMU and 15 μM atrazine, solution, to eliminatephotosynthetic oxygen evolution. The cultures were then incubated underlow light conditions, in anaerobic jars, containing a nitrogen gasmixture, having approximately 10% hydrogen, 1% carbon dioxide, and 8-10%oxygen, for seven days in order to eliminate the wild-type andundesirable phenotypes. After seven days, the surviving oxygen-tolerantmutant cells were washed in growth medium, and plated on sterile minimalagar. Surviving colonies were counted after growing under illuminationconditions for three weeks.

The addition of oxygen, to the selective pressure, decreased the numberof survivors by 3-4 orders of magnitude because of the inactivation ofoxygen-sensitive hydrogenase. When the cells were cultured in oxygen formore than seven days, the cell density increased once again due toreplication of the oxygen-tolerant survivors.

Screening

An individual 3 mm diameter colony of green algae growing on agarcontains about 1 μg of chlorophyll. We had previously determined thatthis colony can photo-evolve hydrogen, under anaerobic conditions, at amaximum measurable rate of about 80 μmoles of hydrogen per milligram ofchlorophyll per hour. Thus, the theoretical yield of this colony is 4nmoles of hydrogen in the two minutes prior to deactivation of thehydrogenase enzyme, by oxygen, as a result of photosynthesis. We haveconsidered this number to be the minimum desired sensitivity forscreening organisms suspected of having the oxygen-tolerant hydrogenphenotype.

A sterile piece of filter paper was positioned on top of the platedmutant colonies surviving hydrogen-uptake selection pressure, and thehydrogenase was induced by subjecting the colonies to an anaerobicatmosphere for 4 hours. The sensor film, of the invention herein, wasplaced on top of the filter paper and the plates were illuminated by alight source 30, as seen in FIG. 1, to generate hydrogen production, inthe sensitization reaction. Hydrogen-induced, chemochromic sensor colorchange results were recorded as illustrated in the following figures.FIG. 3A shows one agar plate containing several different survivingalgal isolate colonies. The colonies are covered with a piece of filterpaper 15 and the locating grid 35, see also, FIG. 1. FIG. 3B showsplacement of the chemochromic film over the filter paper, and coloniesof algae. In FIG. 3B, the colonies shown are illuminated from the bottomof the agar plate with saturating light causing the induction ofhydrogen production, in the sensitization reaction. In FIG. 3C, thechemochromic sensor film has been removed from the plastic dish and eachdark spot, on the film, corresponds to the grid location covering thehydrogen-producing algal colony causing the color change. Finally, inFIG. 3D, the reversible nature of the color change is demonstrated.Here, the color change spots have disappeared after exposure of the filmfor 5 minutes in air, following the sensitization reaction. Thisdisappearance is confirmation of the fact that hydrogen has evolved foreach surviving colony having the oxygen-tolerant hydrogen phenotype.

These results demonstrate that the chemochromic sensor film is sensitiveenough to detect nanomoles of hydrogen produced by individual coloniesof algae and that this sensor is, therefore, useful to rapidly screen alarge number of colonies for hydrogen production capacity. Preliminaryexperiments have also confirmed a measured quantitative relationshipbetween the intensity of the sensor film color change and the amount ofhydrogen produced.

Colonies that had exhibited a light blue color change, and a dark bluecolor change were raised separately, in liquid culture. The hydrogenaseenzyme was then induced, in each culture, and the initial rates ofhydrogen evolution were measured by amperometric measurement. Theculture which had exhibited the dark blue sensor film color change had ahigher initial rate of hydrogen production compared to cultures whichexhibited the light blue color change. Thus far, after one round ofselection and screening, according to the invention herein, the bestmutant produced hydrogen at a rate of four times that of the WTorganism, and was about three times less sensitive to oxygen.

We claim:
 1. A method for identifying a hydrogen-gas-producing organismcomprising the steps of: (a) isolating the organism on a substrate; (b)providing a sensor film having a first layer comprising a transitionmetal oxide or oxysalt and a second layer comprising ahydrogen-dissociative catalyst metal, the first and second layers havingan inner and an outer surface wherein the inner surface of the secondlayer is deposited on the outer surface of the first layer; (c)positioning the outer surface of the second layer proximal to thesubstrate; and (d) identifying the organism by inducing the evolution ofhydrogen gas, the gas causing a change in the color of the film.
 2. Themethod of claim 1, wherein the transition metal oxide is selected fromthe group consisting of WO₃, Nb₂O₃ and CoMoO₄.
 3. The method of claim 1,wherein the hydrogen dissociative catalyst metal is selected from thegroup consisting of rhodium, platinum, and palladium.
 4. The method ofclaim 1, further comprising providing a fluorinated hydrocarbon polymerbetween the outer surface of the second layer and the substrate, thefluorinated hydrocarbon applied to the outer layer of the hydrogendissociative catalyst metal.
 5. The method of claim 4 wherein thefluorinated hydrocarbon polymer comprises polytetrafluoroethylene. 6.The method of claim 1, further comprising a means for separating thesubstrate from the outer surface of the second layer.
 7. The method ofclaim 6, wherein the separating means comprises filter paper.
 8. Themethod of claim 1, further comprising providing a means for associatinga location of the hydrogen producing organism on the substrate inrelation to the color change on the film.
 9. The method of claim 8,wherein the associating means comprises a grid system comprised of amatrix of cells positioned in a spaced relationship to the substrate, asingle colony of the microbe circumscribed by reference to a cell of thegrid.
 10. The method of claim 1, wherein the color change comprises avariable intensity on the film.
 11. The method of claim 10, wherein thevariable intensity is a function of the hydrogen gas concentrationevolved by the microorganism to be identified.